Staphylococcus aureus (S. aureus; SA) is a bacterium, whose natural reservoir is mucous or wet/salty skin areas like the groin, anus or nose. In addition it inhabits wounds. Overall, the nose is the predominant environment for S. aureus. However, S. aureus can cause illnesses ranging from minor skin infections (such as pimples, boils, and cellulitis) and abscesses, to severe diseases such as pneumonia, meningitis, endocarditis, toxic shock syndrome (TSS), septicemia and Multi Organ Dysfunction Syndrome (MODS). Each year some 500,000 patients in American hospitals contract a staphylococcal infection.
Today, S. aureus has become resistant to many commonly used antibiotics. In the UK, only 2% of all S. aureus isolates are sensitive to penicillin with a similar picture in the rest of the world. The β-lactamase resistant-penicillins (methicillin, oxacillin, cloxacillin and flucloxacillin) were developed to treat penicillin-resistant S. aureus and are still used as first-line treatment. Methicillin was the first antibiotic in this class to be used (it was introduced in 1959), but only two years later, the first case of methicillin-resistant S. aureus (MRSA) was reported in England. MRSA may also be known as oxacillin-resistant Staphylococcus aureus (ORSA) and multiple-resistant Staphylococcus aureus, while non-methicillin resistant strains of S. aureus are sometimes called methicillin-susceptible Staphylococcus aureus (MSSA) if an explicit distinction must be made.
Despite its resistance, MRSA generally remained an uncommon finding even in hospital settings until the 1990's when the MRSA prevalence dramatically increased in hospitals where it is now endemic. Moreover, in the US there are increasing reports of outbreaks of MRSA colonisation and infection through skin contact in locker rooms and gymnasiums, even among healthy populations. MRSA also is becoming a problem in pediatrics (Johnson et al., 2005, J Antimicrob Chemother 56 (3):455-62). As of early 2005, the number of deaths in the United Kingdom attributed to MRSA has been estimated by various sources to lie in the area of 3000 per year (Johnson, supra).
Infections caused by MRSA show a higher lethality rate and more severe symptoms, since treatment is restricted because of the resistance to only few antibiotics. First-line treatment for MRSA is currently glycopeptide antibiotics (vancomycin and teicoplanin). However, there are number of problems with these antibiotics, mainly centered around the need for intravenous administration (there is no oral preparation available), toxicity and the need to monitor drug levels regularly by means of blood tests. There are also concerns that glycopeptide antibiotics do not penetrate very well into infected tissues (this is a particular concern with infections of the brain and meninges and in endocarditis). However, glycopeptides must not be used to treat methicillin-sensitive S. aureus as outcomes are inferior. Taken together, patients infected with MRSA do stay longer in hospital than patients just infected with SA.
Furthermore, unrecognized colonization with MRSA may lead to the distribution of MRSA from one patient to the other by replacing the flora of MRSA-uncolonized patients with MRSA. Colonization is eased by certain risk factors including e.g. previous antibiotic treatment, polymorbidity, and diabetes as well as previous stays in hospital.
From this, it follows that it is highly important to have a safe and reliable tool for the diagnosis and/or detection of MRSA.
Presently, the diagnosis of most skin infections is made by the pattern of symptoms and physical exam findings but it is not usually possible to know whether the infection is caused by Staphylococcus bacteria of any type or another bacterium, like e.g. group A Beta-hemolytic Streptococcus (Streptococcus pyogenes). To make a definitive diagnosis and to confirm that MRSA is the bacteria causing the infection, a culture can be done.
However, diagnosing MRSA using a conventional culture requires 16-72 hours, causing a delay in treatment, significantly impairs patient outcomes and may facilitate outbreaks as well as increase hospital costs.
Phenotypic analyses, such as coagulase type, enterotoxin (SE) type, production of toxic shock syndrome toxin-1 and in vitro antibiotic susceptibility, have been used routinely for MRSA strain typing.
Additionally, genotypic analysis for MRSA strains has been established, providing a more detailed classification than phenotypic analysis. In particular, pulse-field gel electrophoresis (PFGE) using restriction fragments of total genomic DNA of MRSA is an excellent method of characterization.
Alternatively, multilocus sequence typing (MLST) may be used in order to characterize isolates of bacteria by using the sequences of internal fragments of seven housekeeping genes. MLST has been developed and validated for S. aureus (Enright et al., 2002, Proc. Nat. Acad. Sci. U.S.A., 99:7687-7692) and provides a discriminatory method that allows related strains recovered in different countries to be readily identified.
Test kits have been developed in order to detect MRSA (e.g., IDI-MRSA assay, Becton Dickinson, USA) which is an in vitro diagnostic test for the direct detection of nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) to help prevent and control MRSA infections in healthcare settings.
However, this kit has a series of disadvantages which limit its usefulness. First, it does not detect MRSA of Staphylococcal Chromosomal Cassette (SCC) type V, which is important as SCC type V is a previously identified type, especially present in Asia and Australia. Second, its applicability is restricted for a higher number of probes. Third, the preparation of the samples is quite complicated or difficult. Fourth, the concept used for the controls is not state of the art. Moreover, the inhibition rate is quite high and, finally, a numerousness of primers and probes used is required, resulting in a diagnostic tool, which is prone to errors. Additionally, melting curve analysis which may be used by the FRET probe assay allows a higher specificity within the output signal compared to amplification curves.